Ribosomal proteins are vital to multiple aspects of cellular function and survival. Mutations in ribosomal structural proteins or in proteins regulating ribosome assembly severely disrupt hematopoiesis in the bone marrow1,2;however, such mutations have not yet been found to selectively disrupt T-lineage cells. Ablation of the gene encoding the ribosomal structural protein Rpl22 results in a strikingly specific defect in T cell development. This proposal seeks to understand the basis for the lineage restriction of the developmental blockade in Rpl22-deficient (Rpl22-/-) mice. Rpl22 is a ubiquitously expressed RNA binding protein that is a component of the 60S ribosomal subunit but is not essential for global or CAP-dependent translation3, 4. Surprisingly, Rpl22-/- mice are healthy, fertile, and of normal size. However, our laboratory finds that ablation of the Rpl22 gene in mice results in a blockade in thymic development of ?? lineage but not ?? lineage T cells5. We have reported that Rpl22-mediated de-repression of p53 translation occurs specifically within the ?? T cell lineage but not in the closely related Rpl22-/- ?? lineage cells. In this proposal, I will define the basis for the lineage-restricted p53 induction through an analysis of the tumor suppressor phosphatase and tensin homolog deleted from chromosome 10 (PTEN). Preliminary data indicate that PTEN expression in Rpl22-/- DN thymocytes (a mixed population of ?? and ?? precursors) is markedly reduced. Subsetting the DN thymocytes into ?? lineage DN3 and ?? precursors revealed that p53 induction occurred in Rpl22-/- ?? lineage cells that retained PTEN expression, but not in Rpl22-/- ?? lineage cells which had lost PTEN expression. I hypothesize that differences in PTEN levels in Rpl22-/- ?? and ?? lineage cells will influence their sensitivity to Rpl22-deficiency because PTEN is a positive regulator of p53. The experiments proposed within this application will determine the mechanism for differences in PTEN expression between ?? and ?? lineage cells in Rpl22-/- mice via p53 regulation. In specific aim 1 of my proposal, will examine the mechanism of PTEN regulation between Rpl22-/- ?? and ?? lineage T cells. I will investigate the level of PTEN expression in Rpl22-/- ?? and ?? T cell populations and then determine whether Rpl22 deficiency controls PTEN expression at the level of mRNA, protein synthesis, or protein stability. Moreover, based on my findings, I will investigate the mechanistic basis for the effect in part by determining if lineage-specific differences exist in known regulators of PTEN transcription6, 7, translation8, or protein stability9, 10. Given that Rpl22-/- ?? lineage cells retain PTEN expression, and thus are subject to a block in development due to translational de-repression of p53 mRNA means that PTEN ablation should reduce p53 levels and allow development to continue. In Specific Aim 2, I will use loss-of-function approach by deleting PTEN specifically from T lineage cells and analyze the effects on ?? lineage cell development. Together this proposal will provide insight into the mechanisms for ?? T cell resistance to Rpl22 deficiency, and potentially into a novel role for p53 regulation. PUBLIC HEALTH RELEVANCE: The extraribosomal functions of ribosomal proteins, such as in regulating cell death, inflammation, and cell cycle, have far-reaching and biologically important consequences. My research will reveal the basis for ribosomal protein Rpl22-mediated regulation of the tumor suppressor protein PTEN, and potentially determine why Rpl22 deficiency imposes a developmental blockade on a major T lymphocyte lineage. My proposal seeks to further our understanding of Rpl22 in these processes which will potentially lead to therapeutic targets of cancer or inflammation.